This study investigates the impact of intermediate capsids on the potency of recombinant adeno-associated virus (AAV) vectors. AAV vectors are produced in three types of capsids: full, intermediate, and empty. While intermediate and empty capsids are generally considered impurities due to their non-functional nature, they can affect product efficacy and patient safety. The study uses preparative ultracentrifugation to fractionate AAV vectors into enriched populations of full, intermediate, and empty capsids. Analytical methods such as analytical ultracentrifugation (AUC), charge detection mass spectrometry (CDMS), and next-generation sequencing (NGS) are employed to characterize the capsid content and genome composition. The results show that intermediate capsids contribute to the vector genome titer but do not enhance the potency of the AAV product. In vitro and in vivo potency assays reveal that while intermediate capsids are infectious, they do not support transgene expression or biological activity. The study emphasizes the importance of reducing and controlling intermediate capsid levels to ensure the safety and efficacy of AAV products.This study investigates the impact of intermediate capsids on the potency of recombinant adeno-associated virus (AAV) vectors. AAV vectors are produced in three types of capsids: full, intermediate, and empty. While intermediate and empty capsids are generally considered impurities due to their non-functional nature, they can affect product efficacy and patient safety. The study uses preparative ultracentrifugation to fractionate AAV vectors into enriched populations of full, intermediate, and empty capsids. Analytical methods such as analytical ultracentrifugation (AUC), charge detection mass spectrometry (CDMS), and next-generation sequencing (NGS) are employed to characterize the capsid content and genome composition. The results show that intermediate capsids contribute to the vector genome titer but do not enhance the potency of the AAV product. In vitro and in vivo potency assays reveal that while intermediate capsids are infectious, they do not support transgene expression or biological activity. The study emphasizes the importance of reducing and controlling intermediate capsid levels to ensure the safety and efficacy of AAV products.